We intend to examine two factors involved in the regulation of transcription of eukaryotic genes. These include: (1) the function of sequences located 5' to a gene and (2) the physical location of the gene in the chromosome. Recent evidence has shown that sequences immediately adjacent to the 5' end of the coding region of many eukaryotic genes (ie. CCAAT, TATA) affect the rate and specificity of transcriptional initiation. Other results suggest that sequences further upstream from the CCAAT and TATA also influence or modulate the expression of individual genes (ie. sea urchin histone genes). We will test the hypothesis that modulating sequences located several hundred nucleotides from the 5' end of the Drosophila Alcohol dehydrogenase gene modulate the expression of this gene. We will create a series of in vitro deletion mutations at the 5' end of the Drosophila ADH gene utilizing restriction endonucleases and molecular cloning. The function of the specific sequences will be analyzed by microinjection of the modified gene into the nuclei of Xenopus oocytes followed by 5' and 3' S1 nuclease mapping of the transcripts. Previous studies have analyzed genes coding for ubiquitous functions. The results of this study will enable us to determine if modulator sequences exist in a gene coding for a differentiated function and to identify the sequence. This information will be necessary in order to formulate basic principles of gene regulation. We have evidence which suggests that DNA sequences microinjected into fertilized eggs are integrated into the Xenopus genome. The second portion of this proposal will determine the effect of the site of integration of both sea urchin histone genes and the Drosophila ADH gene on their developmental and tissue specific expression. This will be approached by microinjecting Drosophila ADH and sea urchin histone genes into fertilized eggs of Xenopus laevis and analyzing the developmental and tissues specific expression of these genes when integrated into different positions in the genome. We feel that this system is especially well suited for these studies since we have evidence for integration of injected sequences, and have devised a system by which we can produce clones or frogs by nuclear transplantation in which each clone will contain the injected gene integrated in a different chromosomal site. The results of this study will have far reaching implication regarding the nature of gene regulatory mechanisms as well as health related genetic procedures.